Abstract: Many flaviviruses such as Dengue virus, Zika virus, West Nile virus, and Yellow Fever virus cause significant human diseases. However, no clinically approved antiviral therapy is available for treatment of flavivirus infections. Therefore, the development of vaccines and antiviral agents for prevention and treatment of flavivirus infections is a clear public health priority. During infection of the host cell, a single viral polyprotein is synthesized from the viral genome. This polyprotein is cleaved into its component proteins by a combination of host cell proteases and a two-component viral protease, encoded in genes NS2B and NS3. The function of this viral protease is indispensable for virus assembly and replication. The goal of this proposal is to develop high throughput screening strategies to identify and characterize compounds that orthosterically inhibit the function of the critical protease. In Specific Aims 1 and 2 we will develop and perform novel high-throughput screening assays capable of screening large chemical libraries to identify orthosteric inhibitors. In Specific Aim 3 we will evaluate the efficacy of the resulting candidate compounds for their capacity to block protease activity. Lead compounds against the protease will be tested for cellular toxicity, reduction of flavivirus titer in cell culture, and resistant variants. We will ultimately test the best lead compound toxicity and efficacy in live animals for the most potent compounds. This study will generate information on flavivirus inhibitor-enzyme interaction at structurally significant sites and potentially lead to novel classes of flaviviral inhibitors.